High molecular weight forms of human placental lactogen: synthesis in vitro and binding to membrane receptors for lactogenic hormones.

Translation in wheat germ extracts of poly(A)-containing RNA isolated from human term placentas resulted in the synthesis of immunoreactive forms of human placental lactogen (hPL) capable of specific binding to lactogenic receptors. The minor component coelectrophoresed on sodium dodecyl sulfate-polyacrylamide gels with authentic hPL while the major component migrated with an apparent molecular weight about 3000 larger. In addition to this precursor-like molecule, even higher molecular weight forms of hPL were observed under certain conditions: (i) when the cell-free translation products were purified by precipitation with anti-hPL serum followed by dissociation of the immunoprecipitate in guanidine hydrochloride and chromatography of the solubilized material on Sephadex G-150 in the same denaturing buffer, and (ii) when the cell-free reaction mixture was analyzed by direct chromatography on Sephadex G-150 in nondenaturing buffers. Under both sets of conditions 50–75% of the radioactivity was eluted in the column void volume, suggesting it had a molecular weight of 150,000 or more. When the high molecular weight translated product was analyzed by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the radioactive components were identical to authentic hPL and the precursorlike form, suggesting the large forms are aggregates of the smaller forms. Both the very high molecular weight forms, composed primarily of the precursor-like molecule, and the less aggregated products bound to specific lactogenic hormone receptors in rat liver membrane preparations, although the larger forms exchanged less readily with unlabeled hPL than did the monomeric form of the hormone. The aggregated, receptor-bindable cell-free translation product may be similar to high molecular weight lactogens previously described in vivo.     

Limited proteolysis of some egg surface components is an early event following fertilization of the sea urchin, Strongylocentrotus purpuratus.

The surface proteins of eggs from Stronglocentrotus purpuratus were labeled with ¹²⁵I by lactoperoxidase-catalyzed iodination. The eggs were examined after solubilization and disaggregation in sodium dodecyl sulphate (SDS) by electrophoresis on SDS-polyacrylamide slab-gels. Seventy-five percent of the label was found in material with a molecular weight greater than 130,000. About 5% of the radioactivity was excluded from the gels. Upon fertilization, embryos show a redistribution of the radioactively labeled species. There is a decrease in the amount of very high molecular weight material but an increase (35–40%) in material excluded from the gel. In addition, new radioactive bands of lower molecular weight are found. This change of distribution in the radioactive bands is blocked by inclusion of soybean trypsin inhibitor either before or immediately after fertilization, which completely inhibits the cortical granule protease. The disappearance of high molecular weight components is prevented by treatment of the eggs with procaine during fertilization, although the appearance of low molecular weight bands (approximately 20,000 and 30,000) is not completely blocked by procaine treatment. Parthenogenic activation of eggs by butyric acid or partial metabolic activation by ammonia each leads to changes in the egg surface proteins which are similar but not identical to those seen after fertilization. The data suggest that the labeling occurs on the vitelline membrane, plasma membrane and jelly layer. The possible significance of limited proteolysis in fertilization is discussed.     

Cell-cell recognition in yeast. Molecular nature of the sexual agglutinin from Saccharomyces kluyveri 17-cells

A previous study of Saccharomyces kluyveri 17-cell sexual agglutinin (alpha-agglutinin), solubilized by zymolyase (beta-glucanase) digestion of 17-cells and purified by affinity adsorption to immobilized 16-cell agglutinin (alpha-agglutinin), suggested that the major active component was a glycoprotein of 60,000 daltons and that a minor active component of 40,000 daltons was also present, possibly the result of proteolysis (Pierce, M., and Ballou, C. E. (1983) J. Biol. Chem. 258, 3576-3582). We now show that both of these active components are proteolytic fragments of a larger form with a molecular weight of 134,000, and that the latter is produced by proteolysis of a still larger species with a molecular weight of more than 200,000. Washed 17-cell wall fragments were labeled with 125I and digested with purified protease-free beta-1,3-glucanase, and the solubilized alpha-agglutinin was precipitated with antiserum raised against purified agglutinin containing a mixture of the 60,000- and 134,000-dalton forms. Gel electrophoresis in sodium dodecyl sulfate revealed a radioactive material with Mr greater than 200,000 that, on digestion with zymolyase containing an active protease, was converted sequentially to radioactive components with Mr = 134,000, 60,000, and 40,000.     

Characterization of lens capsule collagen: evidence for the presence of two unique chains in molecules derived from major basement membrane structures

A collagen fraction representing two-thirds of the collagenous sequences in bovine lens capsules has been isolated following limited pepsin digestion and purified by DEAE- and carboxymethyl-cellulose chromatography in native form. The denaturation products of this collagen contain two types of components. The more acidic components (C and 50K₁) are, respectively an α-chain-sized collagenous polypeptide and a mixture of smaller molecular weight proteolytic cleavage products of the C chain. The more basic components (80K and 50K₂) represent, respectively, a collagenous polypeptide with an apparent M_r = 80,000 and a mixture of smaller molecular weight components derived through proteolysis of the 80K component. The C chain and 80K components are unique with respect to chromatographic properties, amino acid composition, and cyanogen bromide cleavage products. These data are interpreted to indicate that lens capsule basement membrane collagen molecules collectively contain at least two genetically distinct collagen chains: the C chain representing the collagenous domain of one type of chain and the 80K component representing the major portion of the collagenous domain of a second type of chain, designated the D chain.     

Studies on the mechanism of the conversion of cytosol δ-aminolevulinic acid synthase to the mitochondrial enzyme in the liver of the allylisopropylacetamide-treated rat

δ-Aminolevulinic acid (ALA) synthase was partially purified from liver cytosol fraction of rats treated with allylisopropylacetamide (AIA). The cytosol ALA synthase showed an apparent molecular weight of 320,000. The cytosol ALA synthase of this size dissociates into at least three protein components when subjected to sucrose density gradient centrifugation in the presence of 0.25 m NaCl: one is the catalytically active protein with an s value of about 6.4 or a molecular weight of 110,000, and the other two are catalytically inactive binding proteins showing s values of about 4 and 8, respectively. Recombination of the 6.4 S protein and the 4 S protein yielded a protein complex with an apparent molecular weight of 170,000 and recombination of all three protein components resulted in formation of the original cytosol ALA synthase. The cytosol ALA synthase also loses its binding proteins when treated with various proteases; thus, the enzyme-active protein obtained after papain digestion was very similar, if not identical, to mitochondrial ALA synthase. When treated with trypsin, however, the cytosol ALA synthase was converted to an enzyme showing an apparent molecular weight of 170,000, which probably represents the complex of the mitochondria-type enzyme and the 4 S binding protein. The cytosol ALA synthase tends to aggregate to form a dimer with an apparent molecular weight of 650,000–700,000. The aggregated form of the cytosol ALA synthase was less susceptible to trypsin digestion. Hemin strongly stimulated dimer formation of the cytosol ALA synthase and the aggregate produced by contact with hemin was very tight and did not easily dissociate into its respective protein components by sucrose gradient centrifugation or even after treatment with trypsin. The possible mechanisms of the conversion of cytosol ALA synthase to the mitochondrial enzyme and also of the inhibition by hemin of the intracellular translocation of ALA synthase are discussed.     

The major polypeptides of the nuclear pore complex

Nuclear envelopes of maturing oocytes of various amphibia contain an unusually high number of pore complexes in very close packing. Consequently, nuclear envelopes, which can be manually isolated in great purity, provide a remarkable enrichment of nuclear pore complex material, relative to membranous and other interporous structures. When the polypeptides of nuclear envelopes isolated from oocytes of Xenopus laevis and Triturus alpestris are examined by gel electrophoresis, visualized either by staining with Coomassie blue or by radiofluorography after in vitro reaction with [³H]dansyl chloride, a characteristic pattern is obtained (10 major and 15 minor bands). This polypeptide pattern is radically different from that of the nuclear contents isolated from the same cell. Extraction of the nuclear envelope with high salt concentrations and moderately active detergents such as Triton X-100 results in the removal of membrane material but leaves most of the non-membranous structure of the pore complexes. The dry weight of the pore complex (about 0.2 femtograms) remains essentially unchanged during such extractions as measured by quantitative electron microscopy. The extracted preparations which are highly enriched in nuclear pore complex material contain only two major polypeptide components with apparent molecular weights of 150 000 and 73 000. Components of such an electrophoretic mobility are not present as major bands, if at all, in nuclear contents extracted in the same way. It is concluded that these two polypeptides are the major constituent protein(s) of the oocyte nuclear pore complex and are specific for this structure. When nuclear envelopes are isolated from rat liver and extracted with high salt buffers and Triton X-100 similar bands are predominant, but two additional major components of molecular weights of 78 000 and 66 000 are also recognized. When the rat liver nuclear membranes are further subfractionated material enriched in the 66 000 molecular weight component can be separated from the membrane material, indicating that this is relatively loosely associated material, probably a part of the nuclear matrix. The results suggest that the nuclear pore complex is not only a characteristic ubiquitous structure but also contains similar, if not identical, skeletal proteins that are remarkably resistant to drastic changes of ionic strength as well as to treatments with detergents and thiol reagents.     

Some phytotoxic glycopeptides from Ceratocystis ulmi, the Dutch Elm Disease pathogen

Ceratocystis ulmi, the causal agent of Dutch Elm Disease, produces phytotoxic glycopeptides in culture. A mixture of phytotoxic glycopeptides has been prepared by affinity chromatography on a concanavalin A-Sepharose column and collectively they have been termed the toxin. The polydisperse component that makes up the majority of the toxin (80%) by weight has a molecular weight of about 2.7·10⁵. The large molecular weight component (<5%) elutes at the void volume of a Bio-Gel A50 m column. The other component (15%) appears as a trailing peak on the edge of the major component and has an approximate molecular weight of 7 · 10⁴. The toxin is composed of 38% sugar residues, primarily rhamnose and mannose, and 7% amino acid residues. Methylation analysis coupled with mild acid hydrolysis indicates that the backbone of the polysaccharide portion of the toxin is composed of α-1,6-linked mannosyl residues with a 3-linked terminal rhamnosyl residue linked to C-3 of almost every mannosyl residue. The carbohydrate portion of the molecule is linked to the peptide via O-glycosidic linkages to both threonyl and seryl residues. All three components of the toxin are capable of causing wilt in stem cuttings of American elm.     

Isolation and characterization of agglutinin receptor sites: II. Isolation and partial purification of a surface membrane receptor for wheat germ agglutinin

Four different chemical extraction procedures for the isolation of wheat germ agglutinin receptor sites from L1210 cells are described. Fractionation of the biologically active material on Sephadex G-200 columns in pyridine results in two major peaks, the lower molecular weight fraction having a higher inhibitory activity. Electrophoresis in polyacrylamide sodium dodecyl sulfate gels yields four bands. The most active fraction from Sephadex G-200 has an approximate molecular weight between 40000–60000. A preliminary analysis of the active material indicates the presence of sialic acid, neutral sugars and amino sugars, including N-acetylglucosamine.     

Binding of heparin to antithrombin III: The use of dansyl and rhodamine labels

Low molecular weight heparin of low-anticoagulant activity and high molecular weight heparin of correspondingly high activity were prepared by chromatography on protamine-Sepharose; preparations subjected to limited N-desulfation (5–10% free amino groups) by solvolysis were labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or rhodamine B isothiocyanate (RITC). The fluorescent heparins retained approximately 50% of the original anticoagulant activities. Dansyl-heparin on binding to antithrombin III (ATIII) exhibited a 2.5-fold enhancement of dansyl fluorescence intensity. This effect could be prevented by excess unlabeled heparin. A 7900 molecular weight dansyl-heparin preparation bound to ATIII with a stoichiometry of close to 2:1 and with an apparent association constant for binding (K_a) of 4.9 × 10⁵, m^?1, whereas a 21,600 molecular weight fraction bound at 0.7:1 with the protein and with an apparent K_a = 7.9 × 10⁵, m^?1. When ATIII reacted with a mixture of low molecular weight dansyl-heparin and low molecular weight RITC-heparin, there was enhancement of RITC fluorescence emission when excited at the dansyl excitation maximum; this effect was not observed when either of the labeled heparin species was prepared from high molecular weight material. The results are consistent with the proposal that a single molecule of high molecular weight, high-activity heparin occupies two sites when it binds to ATIII, whereas low molecular weight, low-activity heparin binds to the two sites separately.     

Pepsinogen B: the zymogen of pepsin B

1. Pepsinogen B, the precursor of pepsin B, has been isolated by ion-exchange chromatography and gel filtration from neutral extracts of pig gastric mucosa. The material possesses potential activity against acetyl-l-phenylalanyl-l-di-iodotyrosine and against gelatin but has little, if any, potential activity against haemoglobin. 2. The material appears homogeneous in the ultracentrifuge, but on gel filtration and on electrophoresis in starch gel it is shown to be contaminated with a small amount of material having potential activity against haemoglobin. On electrophoresis in starch gel also the material is shown to contain about equal amounts of two major components, both of which have potential activity against the synthetic substrate. Pepsin B has also been shown to contain two active components by electrophoresis under the same conditions. 3. The zymogen is similar to pepsinogen and pepsinogen C in its molecular weight and general physico-chemical properties, but differs from these zymogens in the nature of its N-terminal residues. It is possible that one of the components contains 1 mole of bound phosphate/mole. 4. The material is activated rapidly at pH2 and more slowly at pH4. At both pH values the kinetics of the activation reaction are complex.     

Production of interleukin 1 by SK hepatoma tumor cells

SK hepatoma cells and SK hepatoma conditioned media contain an 18,000-dalton factor which is pyrogenic, stimulates collagenase and prostaglandin production in skin and synovial fibroblasts, induces bone resorption, and stimulates the proliferation of murine thymocytes. These results are consistent with the finding that this tumor cell line produces interleukin 1 [Doyle, M. V., Brindley, L., Kawasaki, E., & Larrick, J. (1985) Biochem. Biophys. Res. Commun. 130, 768-773] since all these activities have been associated with this cytokine. Greater than 80% of the cellular activity has a molecular weight of 30,000, while in contrast, greater than 80% of the activity in the tumor-conditioned media has a molecular weight of 18,000. When active material from the cells is incubated with trypsin, this high molecular weight material is completely converted into an active 18,000 molecular weight species. The isoelectric point of all active material is always between pI 4.0 and 5.1, regardless of molecular weight. All of these results are consistent with the hypothesis that active, high molecular weight interleukin 1 alpha is first synthesized and stored by the tumor cell. This cytokine is then cleaved by a trypsin-like protease to an active, lower molecular weight species which can be secreted into the media.     

A new form of renin in normal human plasma: “big renin” is a mixture of inactive prorenin and the new active high molecular weight renin

A new form of active renin was separated from inactive prorenin in normal human plasma by a new affinity chromatographic method on a column of Cibacron Blue F3GA-agarose. This active renin has a molecular weight of 54,000, considerably higher than the hitherto recognized active renin of 40,000 dalton in human plasma. The molecular weight of inactive prorenin was 56,000±2,000. Active renin produced from the inactive prorenin by trypsin or pepsin digestion or by acid treatment in $\text{in vitro}$ experiments showed a molecular weight of 54,000±2,000. Active renin with a molecular weight of 40,000 was not found in 6 samples of untreated plasma of normal human subjects nor was it formed by treatment with trypsin, pepsin, or acid pH. It is concluded that a large form of active renin (54,000 dalton) exists in normal human plasma which is distinct from a smaller form and that the activatable “big renin” is a mixture of this active renin and totally inactive prorenin. This explains the absence of molecular weight change during the activation of “big renin”.     

The characterization of the proteins which are secreted by the mucocysts of Tetrahymena thermophila

The ‘mucus’ which is discharged by the ciliated protozoan Tetrahymena thermophila has been isolated. All preparations have been shown to consist of a highly consistent mixture of at least 30 proteins varying in molecular weight between 200 000 and 10 000. In marked contrast to mammalian mucins these preparations have a very low (3% w/v) carbohydrate content with glucosamine being the major sugar. The amino acid analyses show that aspartic and glutamic acids and their amides together with serine and glycine account for 45% of the amino acids.     

Expression of the mitochondrial genome in HeLa cells. XVI. Electrophoretic properties of the products of in vivo and in vitro mitochondrial protein synthesis

The size distribution of the proteins synthesized by isolated HeLa cell mitochondria has been analyzed by polyacrylamide gel electrophoresis and compared to that of the in vivo products of mitochondrial protein synthesis.The electrophoretic pattern of the mitochondrial proteins labeled in vitro with [³H]leucine has a group of partially resolved components migrating in the region corresponding to 12,000 to 25,000 molecular weight, and another group, more abundant, in the range from 40,000 to 55,000 molecular weight. This pattern is very similar, after a two-hour incubation of mitochondria, to that of the proteins labeled in vivo in a 30-minute [³H]leucine pulse.     

A partial characterization of the cyclic nucleotide phosphodiesterases of Drosophila melanogaster

The cyclic nucleotide phosphodiesterases in crude homogenate, soluble material, and particulate preparations of adult Drosophila melanogaster flies, hydrolyze cyclic AMP with nonlinear kinetics. Cyclic GMP is hydrolyzed by the phosphodiesterases in crude homogenate and soluble material with linear kinetics. Physical separation techniques of gel filtration, velocity sedimentation, and ion-exchange chromatography reveal that Drosophila soluble fraction contains two major forms of cyclic nucleotide phosphodiesterase. Form I hydrolyzes both cyclic AMP and cyclic GMP. Inhibition experiments suggest that the hydrolysis of both cyclic nucleotides by Form I occurs at a single active site. The K_m's for hydrolysis of both substrates are about 4 μm. This form has a molecular weight of about 168,000 as estimated by gel nitration. Form II cyclic nucleotide phosphodiesterase is specific for cyclic AMP as substrate. Gel filtration indicates that this form has a molecular weight of about 68,000. The K_m for cyclic AMP is about 2 μm.     

In vitro synthesis of the large subunit of ribulose diphosphate carboxylase on 70 S ribosomes

Polyribosomes isolated from greening barley leaves were active in directing protein synthesis, using soluble components isolated from Escherichia coli. A peptide of 55,000 molecular weight was a major product of translation activity. This peptide was precipitated by antibody to ribulose 1,5-diphosphate carboxylase (RuDPCase) and comigrated with the large subunit of RuDPCase on sodium dodecyl sulfate-polyacrylamide gels. Cyanogen bromide peptides of the peptide of 55,000 molecular weight also corresponded to the peptides prepared from authentic RuDPCase large subunit. The peptides synthesized were shown by sucrose density gradient sedimentation to be largely associated with 70 S ribosomes.     

gamma-Glutamyl transpeptidase from WI-38 fibroblasts: purification and active site modification studies

γ-Glutamyl transpeptidase has been purified to homogeneity from WI-38 human fetal lung fibroblasts, following extraction with Triton X-100 in the absence of added proteases. The specific activity of the purified enzyme is 16 units/mg protein at the optimum of pH 8.0. Although this activity value is low, the WI-38 enzyme is very similar to previously described γ-glutamyl transpeptidases in its molecular properties. The native molecule (apparent molecular weight of 82,000) is composed of one light and one heavy subunit (apparent molecular weights of 20,000 and 62,000, respectively). Papain digestion reduces the native molecular weight to an apparent value of 73,000 by proteolysis of the heavy chain. The known active site modifying agent and glutamine analog 6-diazo-5-oxo-l-nor-leucine, completely inactivates the enzyme, coincident with its stoichiometric incorporation into the light subunit. This inactivation is accelerated by maleate and prevented by S-methylglutathione. The WI-38 γ-glutamyl transpeptidase is also inactivated by the fluorescent alkylating agent, 5-iodoacetamidofluorescein. Selective reaction of this reagent with an active site residue is suggested by prevention of the inactivation by S-methylglutathione, the stoichiometric incorporation of the fluorescein moiety, and the loss of one methionine residue per molecule of protein accompanying inactivation.     

GLIAL VERSUS NEURONAL ORIGIN OF MYELIN PROTEINS AND GLYCOPROTEINS STUDIED BY COMBINED INTRAOCULAR AND INTRACRANIAL LABELLING

Abstract— —The contribution of axonal transport to the production of myelin proteins and glycoproteins was investigated using the double labelling technique of combined intraocular and intracerebral injections in the same animal. Myelin and an axolemma-enriched fraction were isolated from pooled optic nerves, chiasma and optic tracts. Separation by gel electrophoresis showed that typical myelin proteins and glycoproteins were only significantly labelled by intracerebral injection. Intraocular injection labelled high molecular weight proteins other than the major Wolfgram protein and the major myelin glycoprotein. Fifteen days after intraocular injection the label was concentrated in a high molecular weight protein which migrated slightly more slowly than the major Wolfgram protein. The pattern of proteins and glycoproteins in myelin labelled by intraocular injection was very similar to that obtained in the axolemma-enriched fraction by the same route. These results indicate that neuronal metabolism and axonal transport do not contribute significantly to the synthesis of specific myelin proteins and glycoproteins, but suggest that the components of myelin fractions which are labelled by intraocular injection are contaminants of axolemmal origin. One of these glycoproteins may prove a useful marker of axolemma membranes.     

The human LT system. III. Characterization of a high molecular weight LT class (complex) composed of the various smaller MW LT classes and subclasses in association with Ig-like molecules.

Cytotoxic activity (lymphotoxin (LT)) present in supernatants from lectin stimulated human lymphocytes in vitro is composed of a heterogeneous system of biological macromolecules which can be separated into multiple classes and subclasses on the basis of their molecular weight and charge. These studies further characterize a large molecular weight human LT class, termed complex (MW >200,000 d), which elutes in the void volume off Sephadex G-150 or Ultrogel AcA 44. Immunological studies on the complex, employing various rabbit anti-LT class and subclass antisera, revealed this material is a macromolecular assemblage of the smaller MW α, β, γ LT classes and subclasses. Furthermore, the reactivity of this material with anti-human Fab′₂ (IgG) indicates these smaller molecular weight LT components can associate with immunoglobulin or Ig-like molecules. The materials present in the LT complex class appear to be noncovalently associated, since conditions of high ionic strength dissociate certain small MW LT components, while low ionic strength buffers may cause these components to reaggregate with the complex. When subjected to velocity sedimentation on sucrose gradients or gel filtration on Ultrogel AcA 22, LT complex activity elutes as several discrete peaks of activity in the 200,000 to 1,000,000 MW range. These findings suggest the concept that LT molecules can form discrete and specific macromolecular structures which contain the smaller MW LT classes. Moreover, these structures can also associate with immunoglobulin-like molecules to form secondary LT-Ig complexes. This may have important biological significance in explaining how nonspecific cell toxins could play a role in specific or nonspecific cell lytic reactions in vitro.